Our work examines for the first time calcium signaling within nanometer-sized stores in intact living cells in real-time. We first devised a highly sensitive store calcium imaging technique that allows for detection of tiny, brief and extremely confined store calcium depletion signals, named ?calcium blinks?, in heart cells. This approach was further strengthened by a quantitative ultrastructural investigation of the intricate network of cisternae and tubules that comprise the endoplasmic reticulum (ER, or sarcoplasmic reticulum (SR) in muscles). We demonstrate that a calcium blink is a depletion signal from a single junctional SR cisterna (30nm thick by 590 nm diameter disk). Moreover, we show that as calcium sparks (the elementary calcium release events in diverse cells including neurons, muscles as well as some non-excitable cell) arise, calcium blinks develop. While our work has focused on heart muscle cells, the ER/SR is an organelle common to all eukaryotic cells. The classic view is that ER/SR serves as the primary calcium store. However, recent evidence indicates that intra-store calcium plays active signaling roles in capacitative calcium entry, regulation of calcium release and apoptosis. Little has been known previously on intra-SR calcium regulation because of the delicate structure involved, and because of the lack of means to probe calcium inside this membrane-bound internal space with the required sensitivity, resolution and speed. In this regard, both technological and conceptual advances reported here may help with molecular and mechanistic investigations of intra-organelle calcium signaling.